Lrf knockout mice show significantly reduced numbers of LT-HSCs and Flt3⁺ lymphoid progenitors. (A) Representative HSC/progenitor FACS profiles of control (Zbtb7a^Flox/FloxMx1-Cre⁻) and hematopoietic-specific Lrf knockout mice (Zbtb7a^Flox/FloxMx1-Cre⁺). c-Kit–positive cells were purified from total BM cells using c-Kit microbeads and MACS LS columns and stained for surface markers. Arrowhead (bottom row, second from right) indicates CD48^high+ T-cell progenitors within the LSK CD150⁻Flt3⁻ population. Aberrant Sca1^high+ T-cell progenitors within the Lin⁻Flt3⁺Il7Rα⁺ fraction are depicted with an asterisk (second row from bottom, left). LT-HSC: long-term hematopoietic stem cell; CLP: common lymphoid progenitor. (B) Dot graphs of absolute counts of LT-HSCs [LSK (Lin⁻Sca1⁺c-Kit⁺) Il7Rα⁻Flt3⁻CD150⁺CD48⁻VCAM1^dim+] and CLPs (Lin⁻Il7Rα⁺Flt3⁺CD150⁻CD48⁺VCAM1⁻) per 2 legs (2 tibias and 2 femurs). Horizontal black bars: average value; error bars: standard deviation. (C) Proportions of CD34-negative LT-HSCs within the LSK CD150⁺Flt3⁻ population. Horizontal black bars: average value; error bars: standard deviation. (D) Representative FACS profiles of side population analysis in BM cells from control and Lrf knockout mice. (E) Absolute numbers of LT-HSCs in 13.5 dpc fetal liver (FL) from WT (+/+), Lrf heterozygous (+/−) and Lrf knockout (−/−) mice. Horizontal black bars: average value; error bars: standard deviation. (F) Representative FACS profiles for FL T-cell precursors. (G) Dot graphs show proportions (left) and absolute counts (right) of FL T-cell precursors (TER119⁻Gr1⁻c-Kit⁺PIR⁺IL7Rα⁺) in 13.5 dpc FL cells. Horizontal black bars: average value; error bars: standard deviation.